Nucleic acids encoding differentiation inhibitor delta 2

ABSTRACT

Amino acids constituting a physiologically active molecule human delta 2; a gene arrangement thereof; and an antibody thereto. The human delta 2 molecule, at least the amino acid sequence described in SEQ ID NO: 1 of the sequence listing, functions as a chemical useful for proliferation of undifferentiated blood cells and inhibition of differentiation, and hence can be utilized as pharmaceuticals and medical supplies.

This application is a divisional of 09/423,753, filed Dec. 12, 1999, nowU.S. Pat. No. 6,664,098, which is a 371 of PCT/JP98/02104.

This invention relates to a novel bioactive substance.

BACKGROUND OF THE INVENTION

Human blood and lymph contain various types of cells and each cell playsimportant roles. For example, the erythrocyte carries oxygen; plateletshave hemostatic action; and lymphocytes prevent from infection. Thesevarious cells originate from hematopoietic stem cells in the bonemarrow. Recently, it has been clarified that the hematopoietic stemcells are differentiated to various blood cells, osteoclasts and mastcells by stimulation of various cytokines in vivo and environmentalfactors. In the cytokines, there have been found, for example,erythropoietin (EPO) for differentiation to erythrocytes; granulocytecolony-stimulating factor (G-CSF) for differentiation to leukocytes; andplatelet growth factor (mpl ligand) for differentiation tomegakaryocytes which is a platelet producing cells, and the former twohave already been clinically applied.

The undifferentiated blood cells are generally classified into twogroups consisting of blood precursor cells which are destined todifferentiate to specific blood series and hematopoietic stem cellswhich have differentiation ability to all series and self-replicationactivity. The blood precursor cells can be identified by various colonyassays, however identification method for the hematopoietic stem cellshas not been established. In these cells, stem cell factor (SCF),interleukin-3 (IL-3), granulocyte-macrophage colony stimulating factor(GM-CSF),interleukin-6 (IL-6), interleukin-1 (IL-1), granulocyte colonystimulating factor (G-CSF) and oncostatin M have been reported tostimulate cell differentiation and proliferation.

Trials for expansion of hematopoietic stem cells in vitro have beenexamined in order to replace bone marrow transplantation for applyinghematopoietic stem cell transplantation therapy or gene therapy.However, when the hematopoietic stem cells are cultured in the presenceof the above mentioned cytokines, multi-differentiation activities andself-replication activities, which are originally in the position of thehematopoietic stem cells, gradually disappeared and are changed to theblood cell precursors which are only to differentiate to specific seriesafter 5 weeks of cultivation, and multi-differentiation activity whichis one of the specific features of the hematopoietic stem cells, is lost(Wagner et al. Blood 86, 512-523, 1995).

For proliferation of the blood precursor cells, single cytokine is notsufficient to effect, but synergistic action of several cytokines areimportant. Consequently, in order to proliferate the hematopoietic stemcells in maintaining with specific features of the hematopoietic stemcells, it is necessary to add cytokines which suppress differentiationtogether with the cytokines which proliferate and differentiate theundifferentiated blood cells. In general, many cytokines, whichstimulate proliferation or differentiation of cells, are known, butsmall numbers of cytokines, which suppressed cell differentiation, areknown. For example, leukemia inhibitory factor (LIF) has an action ofproliferation of mouse embryonic stem cells without differentiation, butit has no action against the hematopoietic stem cells or blood precursorcells. Transforming growth factor (TGF—β) has suppressive action forproliferation against various cells, but no fixed actions against thehematopoietic stem cells or blood precursor cells.

Not only blood cells but also undifferentiated cells, especially stemcells are thought to be involved in tissue regeneration. Theseregeneration of tissues and proliferation of undifferentiated cells ineach tissue can be applied in various ways by referring to the knownreference (Katsutoshi Yoshizato, Regeneration—a mechanism ofregeneration, 1996, Yodosha Publ. Co.).

Notch is a receptor type membrane protein, which involves in regulationof nerve cells differentiation found in Drosophila. Homologues of theNotch are found in various animal kinds exceeding to the invertebrateand vertebrate including nematode (Lin-12). Xenopus laevis (Xotch),mouse (Motch) or human (TAN-1).

Ligand of the Notch in Drosophila is known. These are Drosophila Delta(Delta) and Drosophila Serrate (Serrate). Notch ligand homologues arefound in various animal kinds as similar to the Notch of receptors(Artavanis-Tsakonas et al., Science 268, 225-232, 1995).

Human Notch homologue, TAN-1 is found widely in the tissues in vivo(Ellisen et al., Cell 66, 649-661, 1991). Three Notch analogousmolecules other than TAN-1 are reported (Artavanis-Tsakonas et al.,Science 268, 225-232, 1995). Expression of TAN-1 was also observed inCD34 positive cells in blood cells by PCR (Polymerase Chain Reaction)(Milner et al., Blood 83, 2057-2062, 1994). However, in relation tohumans, gene and amino acid sequences of human Delta and human Serrate,which are thought to be the Notch ligand, have not been reported asscientific reports in April 1997.

In Drosophila Notch, binding with the ligand was studied andinvestigated in details, and it was found that the Notch can be bound tothe ligand with Ca++ at the binding region, which is a repeated aminoacid sequence No. 11 and No. 12 in the amino acid sequence repeat ofEpidermal Growth Factor (EGF) like repeating (Fehon et al., Cell 61,523-534, 1990, Rebay et al., ibid. 67, 687-699, 1991 and InternationalPublication WO 92/19734). EGF-like repeated sequences are conserved inNotch homologues of the other species. Consequently, the same mechanismin binding with ligand is estimated. An amino acid sequence which iscalled as DSL (Delta-Serrate-Lag-2) near the amino acid terminal, andEGF-like repeated sequence as like in the receptor are conserved in theligand (Artavanis-Tsakonas et al., Science 268, 225-232, 1995).

EGF-like sequence has been found in thrombomodulin (Jackman et al.,Proc. Natl. Acad. Sci. USA 83, 8834-8838, 1986), low density lipoprotein(LDL) receptor (Russell et al., Cell 37, 577-585, 1984), and bloodcoagulating factor (Furie et al., Cell 53, 505-518, 1988), and isthought to play important roles in extracellular coagulation andadhesion.

Recently, the vertebrate homologues of the cloned Drosophila Delta werefound in chicken (C-Delta-1) and Xenopus laevis (X-Delta-1), and it hasreported that X-Delta-1 had acted through Xotch in the generation of theprotoneuron (Henrique et al., Nature 375, 787-790, 1995 and Chitnis etal., ibid. 375, 761-766,1995). Vertebrate homologue of DrosophilaSerrate was found in rat as rat Jagged (Jagged) (Lindsell et al., Cell80, 909-917, 1995). According to the Lindsell et al., mRNA of the ratJagged is detected in the spinal cord of fetal rats. As a result ofcocultivation of a myoblast cell line that is forced excess expressedrat Notch with a rat Jagged expression cell line, suppression ofdifferentiation of the myoblast cell line is found. However, the ratJagged has no action against the myoblast cell line without forcedexpression of the rat Notch.

A hypothesis has been set up so that Notch and its ligand have an actionof differential regulation not only for neuroblasts and myoblasts, butalso for various undifferentiated cells, especially bloodundifferentiated cells. However, as far as clinical applications inhumans, prior known different species such as chicken or Xenopus laevostype Notch ligand have problems with species specificities andantigenicities. Consequently, obtaining prior unknown human Notch ligandis essentially required. The inventor suspected that a molecule havingDSL domain and EGF-like domain which are common to Notch ligandmolecules and a ligand of the human Notch (TAN-1 etc.), which is a humanDelta homologue (hereinafter designates as human Delta) and humanSerrate homologue (hereinafter designates as human Serrate), may befound. In addition, these findings may be a candidate for a drug usefulfor differential regulation of undifferentiated cells.

As a result, in the previous patent application, a gene cloning of threetypes of molecules including human Delta-1, human Serrate-1 and humanSerrate-2 molecules as the human Notch ligand molecules was made, and itwas found that these molecules have an action on blood undifferentiatedcells. (Refer to WO 97/19172 Differentiation-suppressive polypeptide andWO 98/02458 Differentiation-inhibitor). As for the human Notch ligandmolecule, according to the recent report, partial gene and partial aminoacid sequences of the human Delta-1 like molecule, which are, however,incomplete with respect to the specification and disclosure of thefull-length sequence, have been disclosed in the InternationalPublication WO 97/01571. Further, WO 96/27610 discloses total lengthgene and total length amino acid sequences of human Serrate-1(humanJagged-1). Also, WO 96/27610 discloses partial length gene andpartial length amino acid sequences of human Serate-2 (human Jagged-2).This gene sequence might have erroneous sequences and this gene sequencegenerates frame shift, which results completely different amino acidsequence of our WO 98/02458, Differentiation-inhibitor. In addition, thesaid prior arts did not disclose gene cloning of amino terminals.Consequently, the gene sequences and amino acid sequences areincomplete. As a result of searching the gene sequence database,Genebank Release 98 (December 1996), there are four entries about humanSerrate-1, i.e. Registered No. HSU61276, HSU3936, HSU77720 and HSU77914,however no other human Notch ligand molecules are found in the saiddatabase.

The present invention elucidates the gene sequence and amino acidsequence of novel Notch ligand molecules. Novel Notch ligand moleculesand novel therapeutic uses for these molecules are also provided.

In order to search novel human Notch ligands, cross hybridization usingthe human Delta-1 gene was performed.

To obtain the human Delta-1 gene, methods used in the referentialexamples 1 and 2, and WO 97/19172 can be applied. Transformed cells, inwhich a vector pUCDL-1 containing cDNA coding total amino acid sequenceof human Delta-1, i.e. DNA containing sequence from No. 179 to No. 2347in SEQ ID NO: 8, is inserted into E. coli JM109, have been deposited inthe National Institute of Bioscience and Human-Technology, Agency ofIndustrial Science and Technology, Ministry of International Trade andIndustry, in Higashi 1-1-3, Tsukuba, Ibaragi-ken, Japan as permanentculture collection E. coli: JM109-pUCDL-1F. Date of deposition was Oct.28, 1996 as deposition No. FERM BP-5728.

Various lengths of partial genes of this human Delta-1 gene wereprepared. Using these partial lengths of genes as probes, numerous cDNAlibraries were screened under various hybridization conditions todetermine novel Notch ligands by cross hybridization methods.

As a result of extensive studies, the isolation of cDNA coding aminoacid sequences of novel human Delta-2 have been achieved, a novelmolecule having DSL domain common to Notch ligand molecules from humanfetal lung cDNA library, and have prepared the expression systems ofprotein having various forms using the cDNA. Also we have establishedpurification methods of the proteins which were purified and isolated.

Amino acid sequences of novel human Delta-2 are shown in the sequencelistings, SEQ ID NO: 1-3. DNA sequence coding these sequences is shownin the sequence listing, SEQ ID NO: 4.

Physiological actions of the these prepared proteins were searched byusing many types of cells, for example nerve undifferentiated cells,preadipocytes, hepatocytes, myoblasts, skin undifferentiated cells,blood undifferentiated cells and immune undifferentiated cells. As aresult, it has been found that novel human Delta-2 had adifferentiation-suppressive action against undifferentiated blood cells,and had a physiological action to maintain an undifferentiated state.Further, it has been found that the molecule has growth suppressiveaction against vascular endothelial cells.

No significant toxic actions were noted in the toxicity studies on mice,and useful pharmaceutical effects were suggested. Consequently, thepharmaceutical preparations containing the molecule of the presentinvention, medium containing the molecule of the present invention, andthe device immobilized with the molecule of the present invention arenovel drugs and medical materials which can maintain the bloodundifferentiated cells in the undifferentiated condition. Antibodyagainst human Delta-2 is prepared by using antigen of the said humanDelta-2, and purification method of the said antibodies is established.The present invention has completed accordingly.

The present invention relates to a polypeptide comprising at least aminoacid sequence of SEQ ID NO: 1 of the sequence listing, a polypeptidecomprising at least amino acid sequence of SEQ ID NO: 2 of the sequencelisting, and a polypeptide comprising at least amino acid sequence ofSEQ ID NO: 3 of the sequence listing. The present invention also relatesto the said polypeptides having differentiation suppressive actionagainst undifferentiated cells, the said polypeptides in which theundifferentiated cells are undifferentiated cells except for those ofbrain and nervous system or muscular system, the said polypeptides inwhich the undifferentiated cells are undifferentiated blood cells, andthe said polypeptides acting on vascular cells. The present inventionalso relates to a pharmaceutical composition comprising the saidpolypeptides, and the said pharmaceutical composition havingdifferentiation suppressive action against cells, the saidpharmaceutical composition in which the cells are undifferentiated bloodcells, and the said pharmaceutical composition having regulatory actionagainst vascular cells. The present invention further relates to a cellculture medium comprising the said polypeptides, the cell culture mediumin which the cell is undifferentiated blood cell, and a material havingimmobilized thereto the polypeptide. Further, the present inventionrelates to a method for culturing cells using the cell culture medium orthe material, and the method in which the cells are undifferentiatedblood cells.

The present invention further relates to a DNA coding at least an aminoacid sequence of the sequence listing, SEQ ID NO: 1, said DNA coding atleast an amino acid sequence of the sequence listing, SEQ ID NO: 2, theDNA coding at least an amino acid sequence of the sequence listing, SEQID NO: 3, the DNA having a base sequence from 355 to 927 of the sequencelisting, SEQ ID NO: 4, the DNA having base sequence from 355 to 1854 ofthe sequence listing, SEQ ID NO: 4 and the DNA having base sequence from355 to 2331 of the sequence listing SEQ ID NO: 4. The present inventionstill further relates to a recombinant DNA comprising a DNA selectedfrom the group consisting of the DNAs having ligated to a vector DNAwhich can express said DNA in the host cell, a cell transformed by therecombinant DNA, a method for culturing human cells with the said cells,and a process for production of said polypeptide by culturing the saidcells and isolating the compound produced in the cultured mass. Thepresent invention still more further relates to an antibody specificallyrecognizing a polypeptide having an amino acid sequence of the sequencelisting, SEQ ID NO: 3.

DETAILED DESCRIPTION OF THE INVENTION

Preparation of cDNA necessary for gene manipulation, expression analysisby Northern blotting, screening by hybridization, preparation ofrecombinant DNA, determination of DNA base sequence and preparation ofcDNA library, all of which are series of molecular biologicalexperiments, can be performed according to a description of theconventional textbook for the experiments. The above conventionaltextbook of the experiments is, for example, Maniatis et al. ed.Molecular Cloning, A laboratory manual, 1989, Eds., Sambrook, J.,Fritsch, E. F. and Maniatis, T., Cold Spring Harbor Laboratory Press.

A polypeptide of the present invention has at least polypeptides in thesequence listing SEQ ID NO: 1-3. A mutant and allele, which naturallyoccur in the nature, are included in the polypeptide of the presentinvention unless the polypeptides of the sequence listing, SEQ ID NO:1-3 loses their properties. Modification and substitution of amino acidsare described in details in the patent application by the name of Bennttet al. (National Unexam. Publ. WO 96/2645) and can be prepared accordingto the description thereof.

A DNA sequence coding polypeptides of the sequence listing, SEQ ID NO:1-3 is shown in the sequence listing, SEQ ID NO: 4, together with theiramino acid sequences. In these DNA sequences, even if amino acid levelmutation is not generated, naturally isolated chromosomal DNA or cDNAthereof may have a possibility to mutate in the DNA base sequence as aresult of degeneracy of genetic code without changing amino acidsequence coded by the DNA. A 5′-untranslated region and 3′-untranslatedregion do not involve in amino acid sequence determination of thepolypeptide, so DNA sequences of these regions are easily mutated. Thebase sequence obtained by these regeneracies of genetic codes isincluded in the DNA of the present invention.

Undifferentiated cells in the present invention are defined as cells,which can growth by specific stimulation, and cells, which can bedifferentiated to the cells having specific functions as a result of thespecific stimulation. These include undifferentiated cells of the skintissues, undifferentiated cells of the brain and nervous systems,undifferentiated cells of the muscular systems and undifferentiatedcells of the blood cells. These cells include the cell ofself-replication activity which is called as stem cells, and the cellhaving an ability to generate the cells of these lines. Thedifferentiation-suppressive action means suppressive action forautonomous or heteronomous differentiation of the undifferentiatedcells, and is an action for maintaining undifferentiated condition. Thebrain and nervous undifferentiated cells can be defined as cells havingability to differentiate to the cells of the brain or nerve havingspecific functions by specific stimulation. The undifferentiated cellsof the muscular systems can be defined as cells having ability todifferentiate to the muscular cells having specific functions byspecific stimulation. The blood undifferentiated cells in the presentinvention can be defined as cell groups consisting of the bloodprecursor cells which are differentiated to the specific blood seriesidentified by blood colony assay, and hematopoietic stem cells havingdifferentiation to every series and self-replication activities.Further, in the present invention, vascular cells is defined as generalnomination for cells constituting blood vessels, in which vascularendothelial cells is major constituting cells.

The amino acid sequence in the sequence listing, SEQ ID NO: 1 is asequence of the active center of the novel human Delta-2 of the presentinvention, from which the signal peptide is deleted, i.e. amino acidsequence from the amino terminal to DSL domain, and corresponds to anamino acid No. 1 to 191 in SEQ ID NO: 3 of the matured full lengthaminoacid sequence of the novel human Delta-2 of the present invention.

The amino acid sequence in SEQ ID NO: 2 is amino acid sequence ofextracellular domain of the novel human Delta-2 of the presentinvention, from which the signal peptide is deleted, and corresponds toan amino acid No. 1 to 500 in SEQ ID NO: 3 of the matured full lengthamino acid sequence of the novel human Delta-2 of the present invention.

The amino acid sequence of SEQ ID NO: 3 is the matured full length aminoacid sequence of the novel human Delta-2 of the present invention.

The sequence of SEQ ID NO: 4 is cDNA sequence and total amino acidsequence of the novel human Delta-2 of the present invention, whichcorresponds to the coding region of the said cDNA.

The sequence of SEQ ID NO: 5 is DNA sequence which codes FLAG peptideand amino acid sequence of FLAG peptide used in the present invention.

The sequences of SEQ ID NOs: 6 and 7 are DNA sequences of primers usedin referential example 1.

The sequence of SEQ ID NO: 8 is the cDNA sequence and total amino acidsequence of human Delta-1 used in the present invention.

The sequences of SEQ ID NOs: 9, 10, 12 and 13 are DNA sequences ofprimers used in example 1.

The sequence of SEQ ID NO: 11 is a DNA sequence of a probe used inexample 1.

The sequence of SEQ ID NO: 14 is a DNA sequence of a probe used inexamples 1 and 2.

The sequences of SEQ ID NOs: 15 to NO: 24 are DNA sequences of primersused in example 3.

The left and right ends of the amino acid sequences in the sequencelistings indicate amino terminal (hereinafter designates as N-terminal)and carboxyl terminal (hereinafter designates as C-terminal),respectively, and the left and right ends of the nucleotide sequencesare 5′-terminal and 3′-terminal, respectively.

Cloning of unknown human Notch ligand gene can be performed by thefollowing method. During the evolution of the organisms, a part of aminoacids sequences and gene sequences of the human Notch ligand isconserved. Cloning can be theoretically possible by using the otherNotch legand molecule as a probe. However, in such the crosshybridization, there are many problems, for example, what part ispreferable for the probe or how to set up condition for hybridization,and are not so simple. Further, since the cross hybridization processtends to make cloning many numbers of similar genes simultaneously, ittakes much times for gene sequence analysis, consequently identificationof the objective molecules from the cloned genes is quite difficult.

More than 10 gene fragments have been prepared from the human Delta-1gene. By using said probes, screenings of cDNA libraries which originatefrom more than ten different organs were performed under numeroushybridization conditions and washing conditions. A novel Delta likemolecule has been sought after.

In the plaque hybridization, clones can be obtained by isotope labelingand non-isotope labeling with the probe. Isotope labeling can beperformed by, for example, terminal labeling by using [32P]γ-ATP and T4polynucleotide kinase, or other labeling methods such as nicktranslation or primer extension method can be applied.

As a result, in example 1, a screening of a human fetal lung cDNAlibrary was prepared using a partial gene of the full length gene of thehuman Delta-1 as shown in SEQ ID NO: 8 in the sequence listing, i.e.gene sequence shown in SEQ ID NO: 11 in the sequence listing, as aprobe. As a result of the first screening, about 120 positive plaqueswere isolated, and in the second screening, about 80 positive plaqueswere cloned, then gene sequences of these clones were determined. Mostof these cloned genes were the human Delta-1 gene used as a probe. Amongthem, five clones were found as the novel human Delta-2 gene, which issimilar to the human Delta-1 gene, and the objective novel Notch ligandmolecule was found. these cloned genes were the human Delta-1 gene usedas a probe. Among them, five clones were found as the novel humanDelta-2 gene, which is similar to the human Delta-1 gene, and objectivenovel Notch ligand molecule was found.

Among the above five clones, since there were no signal sequence and noamino terminal sequence, a new probe having a gene sequence as shown inSEQ ID NO: 14 was prepared in order to obtain the full length gene.Further, a screening of said human fetal lung cDNA library was repeatedwith this probe. As a result, cloning of the cDNA coding for the fulllength of gene was accomplished.

This sequence was compared with the databse (Genbank Release 89,December 1996), and found that these were novel sequence.

Examples of plasmids integrated with cDNA are, for (example, other thanpBluescript KS described in example 1, E. coli originated pBR322, pUC18,pUC19, pUC118 and pUC119 (Takara Shuzo Co. Japan), but the otherplasmids can be used, if they can replicate and proliferate in the hostcells. Examples of phage vectors integrated with cDNA are, for example,λgt10 and λgt11, but the other vectors can be used, if they can growthin the host cells. The thus obtained plasmids are transduced intosuitable host cells such as genus Escherichia and genus Bacillus usingcalcium chloride method. Examples of the above genus Escherichia areEscherichia coli K12HB101, MC1061, LE392 and JM109. Example of the abovegenus Bacillus is Bacillus subtilis MI114. Phage vector can beintroduced into the proliferated E. coli by the in vitro packagingmethod (Enquist and Sternberg, Meth. Enzymol., 68, 281-, 1979).

The said amino acid sequence was analyzed hydrophobic part andhydrophilic part from amino acid sequence according to the method ofKyte-Doolittle (J. Mol. Biol. 151: 105, 1982). As a result, the novelhuman Delta-2 of the present invention is expressed on cells as cellmembrane protein having one transmembrane domain.

According to an analysis of the amino acid sequence of the novel humanDelta-2, an amino acid sequence of a precursor of the novel humanDelta-2 consists of a 685 amino acid residue shown in the sequencelisting, SEQ ID NO: 4, and the signal peptide domain is estimated tocorrespond to the amino acid sequence of 26 amino acids residue from No.−26 methionine to No. −1 glycine of the sequence listing; extracellulardomain: 500 amino acids residue from No. 1 serine to No. 500 serine;transmembrane domain: 26 amino acids residue from No. 501 phenylalanineto No. 526 valine; and intracellular domain: 133 amino acids residuefrom No. 527 arginine to No. 659 valine. The domain construction isestimated from the amino acid sequences, and an actual presence forms onthe cells. Furthermore, a solution may be possible that differs from theabove structure, and structural amino acid sequence of each domainhereinabove as defined by possibly changing 5 to 10 amino acids of thesequence.

N-terminal amino acid sequence of the human Delta-2 polypeptide, whichis expressed in COS-7 cells, produced and purified as described inexample 5, has at least the amino acid sequence started from No, 1serine in the sequence listing, SEQ ID NO: 2. Similarly, identicalN-terminal can be expected, if the said peptide is expressed in theother animal cells.

According to a comparison in the full length amino acid sequence of thenovel human Delta-2 of the present invention with other Notch ligandmolecules, which has been reported by April 1997, the homology withhuman Delta-1 (amino acid sequence of SEQ ID NO: 8 in the sequencelisting) as a molecule originated from human is 48.5%; with Serrate-1(Genbank HSU61276 and HSU73939) is 40.3%; and with human Serrate-2(Japanese Patent Appln. No. 8-18622, Differentiation-suppressivepolypeptide in the name of the present inventors) is 42,7%. Thehomologies with Delta of other vertebrates are: mouse Delta-1 (D111,Genbank MMDELTA1) 48.7%: flog Delta-1 (Genbank XELXDEL) 47.0%; flogDelta-2 (Genbank XLU70843) 49.7%, and chicken Delta-1 (Genbank GGU26590)47.9%

As the result, the human Delta-2 of the present invention is a novelmolecule, which has never been reported in humans or in other biologicalhomologues, and is a novel substance having an amino acid sequencedifferent from these substances, and is a novel substance which has beenelucidated for the first time by the present inventor. Moreover, nopolypeptide having the same amino acid sequence as the novel humanDelta-2 has been found by a homology search in other organisms.

The homologues of Notch ligand have an evolutionary conserved commonsequence, i.e. a DSL sequence and repeated EGF-like sequence. As aresult of a comparison with the novel human Delta-2 and human Delta-1,these conserved amino acid sequences of the novel human Delta-2 areestimated.

Namely, DSL sequence corresponds to 43 amino acids residue from No. 149cystine to No. 191 cystine of the amino acid sequence in the sequencelisting, SEQ ID NO. 4. EGF-like sequence exists with 8 repeats wherein,in the amino acid sequence in the sequence listing, SEQ ID NO: 4, thefirst EGF-like sequence corresponds to the sequence from No. 196 cystineto No. 224 cystine: the second EGF-like sequence corresponds to thesequence from No. 227 cystine to No. 225 cystine; the third EGF-likesequence corresponds to the sequence from No. 262 to No. 295 cystine:the fourth EGF-like sequence corresponds to the sequence from No. 302cysteine to No. 333 cysteine; the fifth EGF-like sequence corresponds tothe sequence from No. 340 cysteine to No. 373 cysteine; the sixthEGF-like sequence corresponds to the sequence from No. 380 cysteine toNo. 411 cysteine; the seventh EGF-like sequence corresponds to thesequence from No. 418 cysteine to No. 449 cysteine; and the eighthEGF-like sequence corresponds to the sequence from No. 458 cysteine toNo. 491 cysteine.

A part of sugar chain attached is estimated from amino acid sequence ofthe novel human Delta-2 may be No. 82, 157, 179 and 367 asparagineresidue in the sequence listing, SEQ ID NO: 4 as a possible binding siteof N-glycoside bonding for N-acetyl-D-glucosamine. O-glycoside bond ofN-acetyl-D-galactosamineis estimated to be a serine or threonine residuerich part. Protein bound with sugar chain is generally thought to bestable in vivo and to have strong physiological activity. Consequently,in the amino acid sequence of polypeptide having sequence of thesequence listing SEQ ID NO: 1, 2 or 3, polypeptides having N-glucosideor O-glucoside bond with sugar chain of N-acetyl-D-glucosamine orN-acetyl-D-galactosamine is included in the present invention. As shownin example 5, if the human Delta-2 of the present invention is expressedby gene inserted COS-7 cell, at least more than two forms are expresseddue to attached sugar chain as the proteins having different molecularweight.

As a result of studies on binding of Drosophila Notch and its ligand,amino acid region necessary for binding with ligand of Drosophila Notchwith the Notch is from N-terminal to DSL sequence of the maturedprotein, in which signal peptide is removed (International PublicationWO 92/19734). Further, similarly, studies using Nematode by Fitzgeraldand Greenwald (Development, 121, 4275-4282, 1995) clearly indicate thatNotch ligand like molecule APX-1 required for Notch like receptoractivation is sufficient from amino terminal to DSL domain in the fulllength sequence.

This fact indicates that a domain necessary for expression of ligandaction of human Notch ligand molecule is at least the DSL domain, i.e. adomain containing amino acid sequence from No. 149 cycteine to No. 191cycteine in the sequence listing, SEQ ID NO: 1, and a domain at leastnecessary for expression of ligand action of human Delta-2 is novelamino acid sequence shown in the sequence listing, SEQ ID NO: 1, furthera domain at least necessary for expression of ligand action of humanDelta-2 is novel amino acid sequence shown in the sequence listing, SEQID NO: 2.

As shown in example 2, mRNA of the human Delta-2 can be detected byusing DNA coding a part or full of gene sequence in the sequencelisting, SEQ ID NO: 4. For example, a method for detection of expressionof these genes can be achieved by applying with hybridization or PCR byusing complementary nucleic acids of above 12mer or above 16mer,preferably above 20mer having nucleic acid sequence of a part ofswquence in the sequence listing, SEQ ID NO: 4, i.e. antisense DNA orantisense RNA, its methylated, methylphosphated, deaminated orthiophosphated derivatives. Using the same method, detection ofhomologues of the gene of other organisms such as mice or gene cloningcan be achieved. Further cloning of genes in the genome including humanscan be made. Using these genes cloned by such like methods, furtherdetailed functions of the human Delta-2 can be clarified. For example,using the recent gene manipulation techniques, every methods includingtransgenic mouse, gene targeting mouse or double knockout mouse in whichgenes relating to the gene of the present invention are inactivated, canbe applied. If abnormalities in the genome of the present gene arefound, application to gene diagnosis and gene therapy can be made.

A transformant in which vector pBSDL-2, which contains cDNA coding totalamino acid sequence of the novel human Delta-2 of the present invention,is transformed into E. coli JM109, has been deposited in the NationalInstitute of Bioscience and Human-Technology, Agency of IndustrialScience and Technology, Ministry of International Trade and Industry, of1-1-3, Higashi, Tsukuba-shi, Ibaragi-ken, Japan, as E. coli:JM109-pBSDL-2. Date of deposit was May 9, 1997, and deposition No. isFBRM BP-5941.

Process for production of the novel human Delta-2 polypeptide can beperformed, as shown in example 3, by using expression vector pcDNA 3.Production and purification of various forms of the novel human Delta-2polypeptide using cDNA, which codes amino acid sequence of the novelhuman Delta-2 isolated by the above method are known in the references(Kriegler, Gene Transfer and Expression-A Laboratory Manual, StocktonPress, 1990 and Yokota et al. Biomanual Series 4, Gene Transfer andExpression and Analysis, Yodosha Co., 1994). A cDNA coding the aminoacid sequence of the isolated said human Delta-2 is ligated topreferable expression vector and it is produced in the host cells ofeukaryotic cells such as animal cells and insect cells or prokaryoticcells such as bacteria.

In the expression of the novel human Delta-2 of the present invention,DNA coding polypeptide of the present invention may have the translationinitiation codon in 5′-terminal and translation termination codon in3′-terminal. These translation initiation codon and translationtermination codon can be added by using preferable synthetic DNAadapter. Further for expression of the said DNA, promoter is ligated inthe upstream of the DNA sequence. Examples of vector are plasmidoriginated from the above E. coli, plasmid originated from Bacillus,plasmid originated from yeast or bacteriophage such as λ-phage andanimal viruses such as retrovirus and vaccinia virus.

Examples of promoters used in the present invention are any promoterspreferable for corresponding to the host cells used in gene expression.

In case that the host cell in the transformation is genus Escherichia,tac-promoter, trp-promoter and lac-promoter are preferable, and in caseof host of genus Bacillus, SP01 promoter and SP02 promoter arepreferable, and in case of host of yeast, PGK promoter, GAP promoter andADH promoter are preferable.

In case that the host cell is animal cell, a promoter originated fromSV40, promoter of retrovirus, metallothionein promoter and heat shockpromoter can be applied.

Expression of the polypeptide of the present invention can be made byusing only DNA coding the amino acid sequence of the sequence listing,SEQ ID NO: 1, 2 or 3. However, the protein added with specific functioncan be produced by using DNA, to which added cDNA coding the knownantigen epitope for easier detection of the produced polypeptide oradded cDNA coding the immunoglobulin Fc for forming multimer.

As shown in Example 3, we have prepared expression vectors, whichexpress extracellular proteins of the novel human Delta-2, as follow:

1) DNA coding the amino acids from No. 1 to 500 in amino acid sequencein the sequence listing, SEQ ID NO: 2;

2) DNA coding chimera protein, to which added polypeptide having 8 aminoacids, i.e. an amino acid sequence consisting of Asp Tyr Lys Asp Asp AspAsp Lys (hereinafter designates FLAG sequence, an example of DNAsequence coding the same is shown in the sequence listing, SEQ ID NO:5), in the C-terminal of the amino acids from No. 1 to 500 in amino acidsequence in the sequence listing, SEQ ID NO: 2; and

3) DNA coding chimera protein, to which added Fc sequence from the hingeregion of human IgG1 in the C-terminal of the amino acids from No. 1 to500 in amino acid sequence in the sequence listing, SEQ ID NO: 2, isligated to each separately with the expression vector pcDNA 3(INVITROGEN Corp., U.S.A.), then the expression vector expressing theextracellular region of the novel human Delta-2 is prepared.

The expression vector for expression of full length of the novel humanDelta-2 can be prepared as follows:

4) DNA coding amino acids from No. 1 to 659 in the sequence listing, SEQID NO: 3 and

5) DNA coding chimera protein, to which added polypeptide having FLAGsequence in the C-terminal of amino acids from No. 1 to 659 in thesequence listing, SEQ ID NO: 3 are ligated individually with theexpression vector pcDNA to prepare the expression vector, which canexpress full length of the novel human Delta-2.

The transformants are prepared by using these expression plasmidscontaining DNA coding the thus constructed said human Delta-2.

Examples of the host are genus Escherichia, genus Bacillus, yeast andanimal cells. Examples of animal cells are simian cell COS-7 and Vero,Chinese hamster cell CHO and silk worm cell SF9.

As shown in Example 4, the expression vectors of the above 1)-5) aretransduced individually; the novel human Delta-2 is expressed in COS-7cell (obtainable from the Institute of Physical and Chemical Research,Cell Development Bank, RCB0539), and the transformants, which aretransformed by these expression plasmids, can be obtained. Further, thenovel human Delta-2 polypeptide can be produced by culturing thetransformants under preferable culture condition in medium by knownculture method.

As shown in Example 5, the novel human Delta-2 polypeptide can beisolated and purified from the above cultured mass, in general, by thefollowing methods.

For extraction of the substance from cultured microbial cells or cells,microbial cells or cells are collected by known methods such ascentrifugation after the cultivation, suspended in preferable buffersolution, disrupted the microbial cells or cells by means ofultrasonication, lysozyme and/or freeze-thawing and collected crudeextract by centrifugation or filtration. The buffer solution may containprotein-denaturing agents such as urea and guanidine hydrochloride orsurface active agents such as Triton-X. In case of secretion in thecultured solution, the cultured mass is separated by the known methodsuch as centrifugation to separate from microbial cells or cells and thesupernatant solution is collected.

The thus obtained novel human Delta-2, which is contained in the cellextracts or cell supernatants, can be purified by known proteinpurification methods. During the purification process, for confirmationof existence of the protein, in case of the fused proteins such as theabove FLAG and human IgGFc, they can be detected by immunoassay usingantibody against known antigen epitope and can be purified. In case ofnot to express as such the fused protein, the antibody in Example 6 canbe used for detection.

Antibodies, which specifically recognize human Delta-2, can be preparedas shown in Example 6. Antibodies can be prepared by the methodsdescribed in the reference (Antibodies a laboratory manual, E. Harlow etal., Cold Spring Harbor Laboratory) or recombinant antibodies expressedin cells by using immunoglobulin genes isolated by gene cloning method.The thus prepared antibodies can be used for purification of the novelhuman Delta-2. The human Delta-2 can be detected and assayed by usingantibodies which recognize specifically novel human Delta-2 as shown inExample 6, and can be used for diagnostic agents for diseasesaccompanied with abnormal differentiation of cells such as malignanttumors.

More useful purification method is the affinity chromatography usingantibody. Antibodies used in this case are antibodies described inExample 6. For fused protein, antibodies against FLAG in the case ofFLAG, and protein G or protein A in the case of human IgGFc as shown inExample 5.

Physiological functions of the thus purified human Delta-2 protein canbe identified by various assay methods, for example, physiologicalactivity assaying methods using cell lines and animals such as mice andrats, assay methods of intracellular signal transduction based onmolecular biological means, binding with Notch receptor etc.

Actions for blood undifferentiated cells have been observed by usingIgG1 chimera protein of novel human Delta-2. As a result, it has beenfound that, as shown in example 7, in undifferentiated umbilical cordderived blood cells, in which the CD34 positive cell fraction isconcentrated, the novel human Delta-2 has suppressive action of colonyforming action against blood undifferentiated cells, which show colonyformation in the presence of cytokines.

Further as shown in example 8, it has been found that by evaluatingLTC-IC (Long-term Culture-Initiating Cells), which are positioned frommost undifferentiated blood stem cells in the human bloodundifferentiated cells, after culturing the undifferentiated umbilicalcord derived blood cells, in which the CD34 positive cell fraction isconcentrated, in the presence of the human Delta-2 with variouscytokines in serum-free medium, the human Delta-2 has an activity tomaintain a number of LTC-IC. Further, example 9 shows that the humanDelta-2 is Bound with human blood undifferentiated cells.

The results indicate that the human Delta-2 suppresses differentiationof blood undifferentiated cells, and this action is obviously effectivefor cells from blood stem cells to colony forming cells. Thesephysiological actions are essential for in vitro proliferation of bloodundifferentiated cells. Especially, cells cultured in the mediumcontaining human Delta-2 are efficient in recovery of suppression ofbone marrow after administration of antitumor agents, accordingly invitro expansion of hemopoietic stem cells may be possible if otherconditions would be completed. Further pharmaceuticals containing thepolypeptide of the present invention have protective and reduced actionsagainst the bone marrow suppressive action due to adverse effects ofantitumor agents.

In these experiments, the LTC-IC maintaining activity and binding actionfor blood cells of the novel human Delta-2 of the present invention arestronger than those of the human Delta-1 (WO 97/19172), which has sameaction as shown by the present inventors.

As shown in example 9, IgG1 chimera protein of the human Delta-2 isbound with CD34 positive blood undifferentiated cells. By this bindingactivity, the polypeptide of the present invention can be used forisolation and detection of cells. Although the isolation method can beperformed by a method using flow cytometer as described in example 9, amethod using materials, to which polypeptide of the present invention isimmobilized, as described in example 11 may be more convenient.Consequently, cell isolation method using polypeptide of the presentinvention is included in the present invention. Further, cell isolationmethod using a material, to which polypeptide of the present inventionis immobilized, and device for cell isolation applied with the saidisolation method is also includedin the present invention. Any cellisolation method using antibodies described in the references isapplicable to these isolation devices and isolation methods. Forexample, Dynabeads of Dynal Corp., Norway, which is a method usingcombination of magnetic beads and antibodies, can be used.

Further, as shown in example 12, IgG1 chimera protein of the novel humanDelta-2 of the present invention has suppressive action againstproliferation of intravascular endothelial cells and has inhibitoryaction against vascularization. Consequently, the polypeptide of thepresent invention can be used as therapeutic agents for diseases anddisease states, which may be cured by suppressing vascularization asproposed by Folkman and Klagsbrun (Science 235, 442-447, 1987). Concreteexamples of use are described in the above reference, and are, forexample, diseases including malignant tumors.

Suppressive action for differentiation of cells in the undifferentiatedcells other than blood cells is expected and stimulating action fortissue regeneration can be expected.

In the pharmaceutical use, polypeptides of the present invention arelyophilized with adding preferable stabilizing agents such as humanserum albumin, and are used in dissolved or suspended condition withdistilled water for injection when it is in use. For example,preparation for injection or infusion at the concentration of 0.1-1000μg/ml may be provided. A mixture of the compound of the presentinvention 1 mg/ml and human serum albumin 1 mg/ml divided in a vialcould maintain activity of the said compound for long term. Forculturing and activating cells in vitro, lyophilized preparation orliquid preparation of the polypeptide of the present invention areprepared and are added to the medium or immobilized in the vessel forculture. Toxicity of the polypeptide of the present invention wastested. Any polypeptide, 10 mg/kg was administered intraperitoneally inmice, but no death of mice was observed.

In vitro physiological activity of the polypeptide of the presentinvention can be evaluated by administering to disease model mice or itsresembled disease rats or monkeys, and examining recovery of physicaland physiological functions and abnormal findings. For example, in caseof searching abnormality in relation to hemopoietic cells. bone marrowsuppressive model mice are prepared by administering 5-FU series ofantitumor agents, and bone marrow cell counts, peripheral blood cellcounts and physiological functions are examined in the administeredgroup or the non administered group of mice. Further, in case ofsearching in vitro cultivation and growth of hemopoieticundifferentiated cells including hemopoietic stem cells, the bone marrowcells of mice are cultured in the groups with or without addition of thecompound of the present invention, and the cultured cells aretransferred into the lethal dose irradiated mice. Result of recovery isobserved with the indications of survival rate and variation of bloodcounts. These results can be extrapolated to the humans, and accordinglyuseful effective data for evaluation of the pharmacological activitiesof the compound of the present invention can be obtained.

Applications of the compound of the present invention forpharmaceuticals include diseases with abnormal differentiation of cells,for example leukemia and malignant tumors. These are cell therapy, whichis performed by culturing human derived cells in vitro with maintainingtheir original functions or adding new functions, and a therapy, whichis performed by regenerating without damaged the functions of theoriginally existed in the tissues by administering the compound of thepresent invention under the regeneration after tissue injury. Amount ofadministration may differ in the type of preparation and is ranged from10 μg/kg to 10 mg/kg.

Further strong physiological activity can be achieved by expression offorming multimer of the polypeptide of the present invention.

Human Delta-2 having multimer structure can be produced by the method ofexpressing chimera protein with human IgG Fc region as described in theexamples 3 and 4 and expressing the multimer having disulfide bond withhinge region of the antibody, or a method expressing chimera protein, inwhich antibody recognition region is expressed in the C-terminal orN-terminal, and reacting with the polypeptide containing extracellularpart of the thus expressed said human Delta-2 and the antibody whichrecognize specifically the antibody recognition region in the C-terminalor N-terminal. In the other methods, a method, in which a fused proteinwith only the hinge region of the antibody is expressed and the dimer isformed by disulfide bond, can be mentioned. The multimer of humanDelta-2 having higher specific activity than the dimer can be obtained.The said multimer is constructed by fused protein which is prepared forexpressing the peptide in the C-terminal, N-terminal or other region.The protein is prepared in the form of forming disulfide bond withouteffecting in any activities of the other human Delta-2. The multimerstructure can also be expressed by arranging one or more peptide, whichis selected from polypeptides containing amino acids sequence of thesequence listing, SEQ ID NO: 1, 2 and 3, with genetic engineering methodin series or in parallel. Other known methods for providing multimerstructure having dimer or more can be applied. Accordingly, the presentinvention includes any polypeptides containing amino acid sequencesdescribed in the sequence listing, SEQ ID NO: 1, 2 and 3 in the form ofdimer or more structure prepared by genetic engineering technique.

Further in the other method, multimerization method using chemicalcross-linker can be mentioned. For example, dimethylsuberimidatedihydrochloride for cross-linking lysine residue,N-(γ-maleimidebutyryloxy) succinimide for cross-linking thiol group ofcysteine residue and glutaraldehyde for cross-linking between aminogroups can be mentioned. The multimer with dimer or more can besynthesized by applying these cross-linking reactions. Accordingly, thepresent invention includes any polypeptides containing amino acidsequences described in the sequence listing, SEQ ID NO: 1, 2 or 3 in theform of dimer or multimer structure prepared by chemical cross-linkingagents.

In application of medical care in which cells are proliferated andactivated in vitro and are returned to the body, human Delta-2 of theform hereinabove can be added directly in the medium, but immobilizationcan also be made. Immobilization method includes applying amino group orcarboxyl group in the peptide, using suitable spacers or the abovementioned cross-linkers, and the polypeptide can be covalently bound tothe culture vessels. In example 11, method for preparation of theimmobilized material and their effect are illustrated. Accordingly, thepresent invention includes any polypeptides containing amino acidsequences described in the sequence listing, SEQ ID NO: 1, 2 or 3 in theform of existing on the solid surface.

Since the natural human Delta-2 is cell membrane proteins,differentiation suppressive action in example 7, 8 and 12 can beexpressed by cocultivating with cells expressing these molecules andblood undifferentiated cells. Consequently, this invention includes amethod for cocultivation of undiferentiated cells with transformed cellsby using DNA coding amino acid sequence in the sequence listing, SEQ IDNO: 1, 2 or 3. An example is illustrated in example 10. Expressed cellmay be simian COS-7 cell or mouse BaIb 3T3 cells as shown in examples,but cells of human origin are preferable, and further expressed cellsmay be any of human in vivo blood cells and somatic cells rather thancell lines. Consequently, the polypeptide can be expressed in vivo byintegrated into vectors for gene therapy. Examples of vectors for genetherapy are retrovirus vector, adenovirus vector or adeno-related virusvector.

This fact suggests that inhibition of binding of the polypeptide havingamino acid sequence in the sequence listing, SEQ ID NO: 1, 2 or 3 tothese receptors can be used for finding out molecules and compounds forstimulating cell differentiation. The methods include binding experimentusing radio isotope, luciferase assay using transcriptional controlfactors, a down stream molecule of the Notch receptor, and simulation onthe computer by X-ray structural analysis. Accordingly, the presentinvention includes a screening method for pharmaceuticals usingpolypeptide in the sequence listing, SEQ ID NO: 1, 2 or 3.

BRIEF EXPLANATION OF THE DRAWINGS

FIG. 1: Northern blotting analysis of expression of the human Delta-2mRNA in human organs.

FIG. 2: Binding of HD2EXIG of the present invention and of HD1EXIG as acontrol to the human origin T cell type cell line Jurkat.

FIG. 3: Binding of HD2EXIG of the present invention and of HD1EXIG as acontrol to CD34 positive cells from human umbilical cord bloodmononuclear cells.

EMBODIMENTS OF THE INVENTION

Following examples illustrate the embodiments of the present invention,but are not construed as limiting these examples.

REFERENTIAL EXAMPLE 1 Cloning of PCR Products Using Human Delta-1 Primerand Determination of Base Sequence

Mixed primers corresponding to amino acid sequence conserved inC-Delta-1 and X-Delta-1, i.e. sense primer DLTS1 (sequence listing, SEQID NO: 6) and antisense primer DLTA2 (sequence listing, SEQ ID NO: 7),were used.

A synthetic oligonucleotide was prepared by using automatic DNAsynthesizer with the principle of immobilized method. The automatic DNAsynthesizer used was 391PCR-MATE of Applied Biosystems Inc., U.S.A.Nucleotide, carrier immobilized with 3′-nucleotide, solution andreagents are used according to the instructions by the same corporation.Oligonucleotide was isolated from the carrier after finishing thedesignated coupling reaction and treating the oligonucleotide carrier,from which protective group of 5′-terminal was removed, withconcentrated liquid ammonia at room temperature for one hour. Forremoving the protective groups of nucleic acid and phosphoric acid, thereactant solution containing nucleic acid was allowed to stand in theconcentrated ammonium solution in the sealed vial at 55° C. for over 14hours. Each oligonucleotide, from which the carrier and protectivegroups were removed, was purified by using OPC cartridge of the AppliedBiosystems Inc., and detritylated by using 2% trifluoroacetic acid.Primer was dissolved in deionized water to set final concentration of100 pmol/μl for PCR after purification.

Amplification of these primers by PCR was performed as follows. Humanfetal brain originated cDNA mixed solution (QUICK-Clone cDNA, CLONTECHInc., U.S.A.) 1 μl was used. 10× buffer solution [500 mM KCl, 100 mMTris-HCl (pH 8.3), 15 mM MgCl₂, 0.01% gelatin] 5 μl, dNTP mixture(Takara Shuzo Co., Japan) 4 μl, sense primer DLTS1 (100 pmol/μl) 5 μlwhich was specific to the above vertebrates and antisense primer DLTA2(100 pmol/μl) 5 μl and TaqDNA polymerase (AmpliTaq, Takara Shuzo Co.,Japan, 5 U/μl) 0.2 μl were added thereto, and finally deionized waterwas added to set up total 50 μl. PCR was performed by 5 cycles of acycle consisting of treatment at 95° C. for 45 seconds, at 42° C. for 45seconds and 72° C. for 2 minutes, further 35 cycles of a cycleconsisting of treatment at 95° C. for 45 seconds, at 50° C. for 45seconds and 72° C. for 2 minutes, and finally allowed to stand at 72° C.for 7 minutes. A part of the PCR products was subjected to 2% agarosegel electrophoresis, stained with ethidium bromide (Nippon Gene Co.,Japan). and observed under ultraviolet light to confirm amplification ofabout 400 bp DNA.

Total amount of PCR product was subjected to electrophoresis with 2%agarose gel prepared by low melting point agarose (GIBCO BRL Inc.,U.S.A.), stained by ethidium bromide, cutting out about 400 bp bands ofPCR products by the Delta primer under the UV light, adding distilledwater of the same volume of the gel, heating at 65° C. for 10 minutes,and completely dissolving the gel. The dissolved gel was centrifuged at15000 rpm for 5 minutes to separate supernatant solution after addingequal volume of TE saturated phenol (Nippon Gene Co., Japan) and thesame separation operation was performed after adding TE saturatedphenol:chloroform (1:1) solution and chloroform. DNA was recovered fromthe final solution by ethanol precipitation.

A vector, pCRII vector (Invitorogen Inc., U.S.A., hereinafter designatesas pCRII) was used. The vector and the above DNA in molar ratio of 1:3were mixed and the DNA was ligated into the vector by using T4 DNAligase (Invitorogen Inc., U.S.A.). The pCRII, to which the DNA wasintegrated, was subjected to gene transduction into E. coli one shotcompetent cells (Invitorogen Inc., U.S.A.) and was spread on thesemi-solid medium plate of L-Broth (Takara Shuzo Co., Japan) containingampicillin (Sigma Corp., U.S.A.) 50 μg/ml and allowed to stand at 37° C.for about 12 hours. The appeared colonies were randomly selected,inoculated in the L-Broth liquid medium 2 ml containing sameconcentration of ampicillin and shake cultured at 37° C. for about 18hours. The cultured bacterial cells were recovered and the plasmid wasseparated by using Wizard Miniprep (Promega Inc., U.S.A.) according tothe attached explanation sheet. The plasmid was digested by restrictionenzyme EcoRI. Integration of the said PCR product was confirmed byincision of about 400 bp DNA. Base sequence of the incorporated DNA inthe confirmed clone was determined by the fluorescent DNA sequencer(Model 373S. Applied System Inc., U.S.A.).

REFERENTIAL EXAMPLE 2 Cloning of Full Length Human Delta-1 and ItsAnalysis

A screening of clones having full length cDNA was performed byhybridization from human placenta origin cDNA library (inserted cDNA inλgt-11, CLONTECH Inc., U.S.A.) in plaques corresponding to 1×10⁶plaques. Generated plaques were transferred onto nylon filter (HybondN+:Amersham Inc., U.S.A.). The transcribed nylon filter was subjected toalkaline treatment [allow to stand for 7 minutes on a filter paperpermeated with a mixture of 1.5 M NaCl and 0.5 M NaOH], followed bytwice neutralizing treatments [allow to stand for 3 minutes on thefilter paper permeated with a mixture of 1.5 M NaCl, 0.5 M Tris-HCl (pH7.2) and 1 mM EDTA]. Subsequently, the filter was shaken for 5 minutesin the 2-fold concentrated SSPE solution [0.36 M NaCl, 0.02 M sodiumphosphate (pH 7.7) and 2 mM EDTA], washed and air-dried. Then the nylonfilter was allowed to stand for 20 minutes on the filter paper, whichwas permeated with 0.4 M NaOH, and was shaken for 5 minutes and washedwith 5-fold concentrated SSPE solution, and was then again air-dried.Screening was conducted in the human Delta-1 probe labeled withradioisotope ³²P using these filters.

DNA probe prepared in referential Example 1 was labeled with ³²P asfollows. A DNA fragment was cut out by EcoRI from pCRII, inserted apurified PCR product (about 400 bp) by human Delta-1 primer anddetermined gene sequence, and was isolated from low melting pointagarose gel. The thus obtained DNA fragment was labeled by DNA labelingkit (Megaprime DNA labeling system: Amersham, U.S.A.). The primersolution 5 μl and deionized water were added to the DNA 25 ng to set uptotal volume of 33 μl, which was treated for 5 minutes in boiling waterbath. Reaction buffer solution 10 μl containing dNTP, α-³²P-dCTP 5 μland T4 DNA polynucleotide kinase solution 2 μl were added thereto,treated at 37° C. for 10 minutes in water bath. Subsequently, themixture was purified by Sephadex column (Quick Spin Column SephadexG-50: Boehringer Mannheim Inc., Germany), then treated for 5 minutes inboiling water bath and ice-cooled for 2 minutes for use.

Hybridization was performed as follows. The prepared filter hereinabovewas immersed into the prehybridization solution consisting of SSPEsolution, a final concentration of each component of which is set at5-fold concentration, 5-fold concentration of Denhardt's solution (WakoPure Chemicals, Japan), 0.5% SDS (sodium dodecyl sulfate, Wako PureChemicals, Japan) and salmon sperm DNA (Sigma, U.S.A.) 10 μg/mldenatured by boiling water, and shaken at 65° C. for 2 hours, then thefilter was immersed into the hybridization solution of the samecomposition of the above prehybridization solution containing³²P-labeled probe by the above described method and shaken at 65° C. for16 hours to perform hybridization.

The filter was immersed into SSPE solution containing 0.1% SDS, shakenat 55° C. and washed twice, further immersed into 10-fold dilution ofSSPE solution containing 0.1% SDS and washed four times at 55° C. Anautoradiography of the washed filter was performed using intensifiedscreen. Clones of strongly exposed part were collected and the plaquesobtained were again spread and screened by the same method hereinbeforeto separate complete single clones.

The thus isolated phage clones were seven clones. Phage of all of theseclones was prepared to about 1×10⁹ pfu, purified the phage DNA usingWithered Lambda Rep (Promega Corp., U.S.A.), digested by restrictionenzyme EcoRI and inserted into pBluescript KS (Stratagene Inc., U.S.A.),which was digested by EcoRI in the same way. DNA sequences of the bothends of these clones were analyzed by DNA sequencer. Three clones of D5,D6 and D7 were the clone containing DNA sequence from No. 1 to 2244 inthe sequence listing, SEQ ID NO: 8. A clone D4 was a clone containingDNA sequence from No. 999 to 2663 in the sequence listing, SEQ ID NO: 8.The clones D5 and D4 prepared the deletion mutant by using kilosequencedeletion kit (Takara Shuzo Co., Japan) according to a description of theattached paper. Full-length cDNA base sequence of the present inventionwas determined using the DNA sequencer from both direction of5′-direction and 3′-direction.

By applying with XhoI site at No. 1214 in DNA sequence in the sequencelisting, SEQ ID NO: 8, D4 and D5 were digested by restriction enzymeXhoI to prepare plasmid pBSDel-1 containing full length of DNA sequencein the sequence listing, SEQ ID NO: 8.

EXAMPLE 1

Cloning of cDNA of the Novel Human Delta-2

Gene cloning of new human Delta homologue was performed using probe ofthe human Delta-1 gene in the sequence listing, SEQ ID NO: 8.

Probe was prepared by PCR using a template of the human Delta-1 fulllength gene pBSDel-1 in the sequence listing, SEQ ID NO: 8 obtained inthe referential example 2, or vector pUCDL-1F, which was deposited inthe above described institution. A sense primer in the sequence listing,SEQ ID NO: 9 (DNA sequence corresponding to a sequence from No. 636 to655 in the sequence listing, SEQ ID NO: 8) and antisense primer in thesequence listing, SEQ ID NO: 10 (a complementary chain of DNA sequencecorresponding to a sequence from No. 1332 to 1351 in the sequencelisting, SEQ ID NO: 8) were used as primers.

A composition of solution for amplification by PCR was a composition ofsolution as described in the referential example 1, except for theprimer and the template. PCR was conducted by the following conditions:a cycle consisting of a cycling at 95° C. for 45 seconds, at 55° C. for45 seconds and at 72° C. for 2 minutes, a cycle of which was performedfor 30 cycles, and finally the mixture was allow to stand at 72° C. for7 minutes. A part of the PCR product was subjected to 1% agarose gelelectrophoresis, stained with ethidium bromide (Nippon Gene Corp.,Japan) and observed by UV light to confirm amplification of about 700 bpcDNA.

The PCR product was cut out from the agarose gel, and the DNA probe waspurified according to a description of the attached sheet in GenecleanII kit (Bio101 Corp., U.S.A.) and diluted with distilled water to 25ng/μl to prepare DNA probe having sequence shown in the sequencelisting, SEQ ID NO: 11.

A human fetal lung cDNA library prepared by λgt10 (Clontech Corp.,U.S.A.) was screened using the above probe according to a methoddescribed in the referential example 2. In the screening, ahybridization was conducted at 55° C. for 16 hours, with washingcondition for immersing into SSC solution containing 1% SDS, shaking atroom temperature and washing six times. Further, a condition immersinginto SSC solution, which was diluted 3-fold containing 0.1%SDS, andwashing at 55° C. was performed.

About 120 million plaques were screened in the first screening under theabove condition, and as a result, about 120 plaques determined aspositive, were subjected to the second screening by the similar methodto separate each phage.

The isolated phage DNA was purified according to a method in thereferential example 2, digested by restriction enzyme EcoRI, ligated topBluescript KS and analyzed DNA sequence by DNA sequencer by the samemethod as described in the referential examaple 1.

About over half number of clones was human Delta-1 having gene sequencein the sequence listing, SEQ ID NO: 8. Among these, five clones, whichwere similar to but have different gene sequence of the human Delta-1,containing new sequences, which were not found in Genbank Release 98 bycomputer software Genetyx CD Ver 36 (Software Development Corp.) werefound. Deletion mutant of DNA sequence of these clones was preparedusing kilosequence deletion kit (Takara Shuzo Co., Japan) according tothe attached instruction manual. Base sequence of full length cDNA ofthe present invention was determined from both direction of 5′-directionand 3′-direction by combining with primer walking method using the saidDNA sequencer.

As a result, clone 4A encodes gene sequence from No. 526 to 3339 of DNAsequence in the sequence listing, SEQ ID NO: 4 (proviso that sequencefrom No. 1296 to 1515 was deleted); clone 22 encodes gene sequence fromNo. 1029 to 3213 of DNA sequence in the sequence listing, SEQ ID NO: 4;clone 65 encodes gene sequence from No. 754 to 3228 of DNA sequence inthe sequence listing, SEQ ID NO: 4; clone 90 encodes gene sequence fromNo. 552 to 2618 of DNA sequence in the sequence listing, SEQ ID NO: 4;and clone 105 encodes gene sequence from No. 669 to 3339 of DNA sequencein the sequence listing, SEQ ID NO: 4 (proviso that in the clone 105,since many insertions of unknown sequences, which were not found in theother clones, were observed in many regions, it might be originated fromprior splicing immature mRNA). In addition, in the clone 65, cytosine ofDNA sequence No. 2294 in the sequence listing, SEQ ID NO: 4 was replacedby thymine. Therefore, serine of DNA sequence No. 647 in the sequencelisting was replaced by threonine.

These clones did not, however. contain gene sequence coding full lengthamino acid sequence, further new probe was prepared and screening wasrepeated.

New probe was prepared by PCR using template of clone 4A which wasisolated in the above process. A probe was prepared by using senseprimer of the sequence listing, SEQ ID NO: 12 (which corresponds to asequence from No. 526 to 545 of DNA sequence in the sequence listing,SEQ ID NO: 4) and antisense primer of the sequence listing, SEQ ID NO:13 (which corresponds to a sequence from No. 918 to 937 of DNA sequencein the sequence listing, SEQ ID NO: 4), and by the same way as describedpreviously. DNA sequence of this probe is shown in the sequence listing,SEQ ID NO: 14.

The screening of the cDNA library using the probe was conducted as samein the first screening procedure. Proviso that the hypridization wasperformed at 65° C. for 16 hours; washing was performed by immersinginto SSC solution containing 0.1% SDS with shaking at room temperature,further immersing into 10-fold diluted SSC solution containing 0.1% SDSwith washing twice at 65° C.

New clones were identified by the screening. As a result of genesequence determination, the clone P having identical sequence with DNAin the sequence listing, SEQ ID NO: 4, and the clone RA having DNAsequence from No. 263 to 2768 in the sequence listing, SEQ ID NO: 4 weredetermined. These two clones were isolated and identified as clonescoding full length novel human Delta-2 protein. A vector containingclone P, which is ligatedin EcoRI site of pBluescript KS, is designatedas pBSDL-2.

EXAMPLE 2

Organs Which Express Novel Human Delta-2

In order to search expression of the new human Delta-2 mRNA, usingfilters of Human Multiple Tissue Northern Blot, Human Multiple TissueNorthern Blot II, Human Multiple Tissue Northern Blot III and HumanFetal Multiple TissueNorthern Blot II (Clontech Corp., U.S.A.), forwhich mRNA was previously transcripted, and using DNA having sequence ofthe sequence listing, SEQ ID NO: 14 described in the example 1 as aprobe, ³²P labeling was performed by the above mentioned procedure usingthe previously mentioned DNA labeling kit (Megaprime DNA labelingsystem: Amersham Corp., U.S.A.), hybridization was conducted accordingto the instruction manual attached to the above filters to detect theexpression. Result is shown in FIG. 1.

As a result, two types of the expressed mRNA having length about 3.8 kband 5 kb were found. The most strong expression in human adult tissuesas expression site was the heart. Relatively strong expressions wereobserved in the placenta, ovary, small intestine, thyroid gland andspinal cord. Obvious expressions were observed in the skeletal muscle,lung, liver, pancreas, thymus, prostate, lymph node, trachea, adrenalgland and bone marrow. Extremely weak expressions were observed in thestomach, spleen and colon. Further no expressions were observed in thebrain, kidney, testis and peripheral blood lymphocyte. Highly expressionwas observed in the fetal lung among the human fetal tissues. Strongexpression is observed in the fetal kidney and weak expressions wereobserved in the fetal liver and fetal brain.

These results indicate that the novel human Delta-2 of the presentinvention might have function relating to the heart in adult. Further,it might have function against vascular cells due to finding ofexpression in the fetal lung.

EXAMPLE 3

Preparation of Expression Vectors of the Novel Human Delta-2

Using the vector pBSDL-2 coding the full length novel human Delta-2 inexample 1, the expression vectors of human Delta-2 proteins mentioned inthe following 1)-5) were prepared. Addition of restriction enzyme sitesand insertion of short gene sequence were performed using ExSitePCR-Based Site-Directed Mutagenesis Kit (Stratagene Inc., U.S.A.)according to the instruction manual.

1) Expression Vector of Secretory Novel Human Delta-2 Protein (HD2EX)

The cDNA coding polypeptide having amino acid sequence from No. 1 to 500in the sequence listing, SEQ ID NO: 2 was ligated into the expressionvector pcDNA3 containing cytomegalovirus promoter and neomycinresistance gene to prepare the expression vector.

For preparation of expression vector of the novel human Delta-2, inorder to make stable expression of gene product, EcoRI site was added inthe 20 bp upper stream for 5′-direction of the initiation codon (genesequence No. 277 in the sequence listing, SEQ ID NO: 4). Using the aboveMutagenesis Kit, a plasmid pBSDL-2, which contained DNA sequence insequence listing, SEQ ID NO: 4 and full length cDNA of human Delta-2were set as the template, and oligonucleotides having gene sequences insequence listing, SEQ ID NO: 15 and SEQ ID NO: 16, ware set as theprimers. Then DNA adding EcoRI site in the 20 bp upper stream for5′-direction was prepared. Hereinafter this plasmid is designated aspBSEco-DL-2.

Next, in order to add the termination codon and restriction enzyme NotIsite after the extracellular C-terminal position, i.e. after DNAsequence coding amino acid sequence up to No. 500 serine residue in thesequence listing, SEQ ID NO: 2, by using the Mutagenesis Kit similarly,using the pBSEco-DL-2 as template and setting oligonucleotides havinggene sequences in the sequence listing, SEQ ID NO: 17 and SEQ ID NO: 18as primers, addition of the termination codon and NotI site wereperformed. Then, the resulted vector was digested by EcoRI and NotI, andabout 1600 bp split gene fragment was ligated in pcDNA3, which wastreated by the same restriction enzymes, to construct the expressionvector. This vector was designated as pHD2EX.

2) Expression Vector of FLAG Chimera Protein of Secretory Novel HumanDelta-2 (HD2EXFLAG)

The cDNA coding chimera protein having amino acid sequence from No. 1 to500 in the sequence listing, SEQ ID NO: 2 and at this C-terminal theFLAG sequence was ligated to the expression vector pcDNA3 to prepare theexpression vector.

Using pBSEco-DL-2 as template, FLAG sequence was added in theextracellular C-terminal, i.e. after serine residue at No. 500 aminoacid in the sequence listing, SEQ ID NO: 2. Then, in order to add thetermination condon and restriction enzyme NotI site, using theMutagenesis Kit, and setting oligonucleotides having gene sequences inthe sequence listing, SEQ ID NO: 19 and SEQ ID NO: 18 as primers, a genecoding FLAG sequence, termination codon and NotI site were added in theC-terminal. This vector was digested by EcoRI and NotI, and about 1600bp split gene fragment was ligated to pcDNA3 treated similarly by therestriction enzyme to construct the expression vector. This vector wasdesignated as pHD2EXFLAG.

3) Expression Vector of IgG1Fc Chimera Protein of Secretory HumanDelta-2 (HD2EXIg)

A gene sequence coding polypeptide having amino acid sequence in thesequence listing, SEQ ID NO: 2 and at this C-terminal an amino acidsequence of Fc region downstream from the hinge region of human IgG1,was ligated into the expression vector pcDNA3 to prepare the expressionvector.

Preparation of fused protein with immunoglobulin Fc protein wasperformed according to the method of Zettlmeissl et al. (Zettlmeissl etal., DNA cell Biol., 9, 347-354, 1990). A gene using genome DNA withintron was applied and the said gene was prepared by using PCR.

Human genome DNA was used as a template and genomic gene sequence codinghuman IgG1Fc region was subjected to PCR by using primers of anoligonucleotide having the sequence in the sequence listing. SEQ ID NO:23 with restriction enzyme BamHI site and an oligonucleotide having thesequence in the sequence listing, SEQ ID NO: 24 with restriction enzymeXbaI site. The thus obtained about 1.4 kbp band was purified anddigested by restriction enzyme BamHI and XbaI (Takara Shuzo Co., Japan),and genes were ligated to pBluescript, which was similarly treated byrestriction enzyme, by using T4 DNA ligase to perform subcloning.

Later, the plasmid DNA was purified and confirmed gene sequence usingsequencer, then the said gene sequence was confirmed as genomic DNA inthe hinge region of heavy chain of the human IgG1. (The sequence isreferred to Kabat et al., Sequence of Immunological Interest, NIHPublication No.91-3242,1991). Namely, this gene has the restrictionenzyme BamHI site at 5′-terminal and XbaI site at 3′-terminal, and iscloned with BamHI site and XbaI site of pBluescript KS. Hereinafter thisplasmid is designated as pBShIgFc.

Using the pBSEco-DL-2 as template, and using the Mutagenesis Kit,restriction enzyme BamHI site was added in the extracellular C-terminal,i.e. after serine at No. 500 in the sequence listing, SEQ ID NO: 3.Furthermore, in order to add restriction enzyme NotI site to thedownstream for ligating DNA coding the above human immunoglobulinIgG1Fc, these sites were added by using oligonucleotides having genesequences in the sequence listing, SEQ ID NO: 20 and SEQ ID NO: 18 andthe Mutagenesis Kit. During this process, in order to not to shift theframe coding amino acid as a result of addition of BamHI site. AGC inthe DNA sequence coding No. 500 serine on the DNA sequence in thesequence listing, SEQ ID NO: 4, was replaced to TCG.

The thus prepared vector was digested by NotI and BamHI, and about 1200bp gene fragment digested and splited from the above pBShIgFc by NotIand BamHI was ligated into the digested vector to prepare finally thevector containing gene fragments coding the objective secretory humanDelta-2 IgG1Fc chimera protein. Finally, this vector was digested byEcoRI and NotI and about 3000 bp split gene fragment was ligated intopcDNA3 which was treated similarly with the restriction enzymes, toconstruct the expression vector. This vector was designated as pHD2EXIg.

4) Expression Vector of Full Length Human Delta-2 Protein (HD2F)

The cDNA coding polypeptide from No. 1 to 659 of amino acid sequence inthe sequence listing, SEQ ID NO: 4, was ligated into the expressionvector pcDNA3 to prepare the expression vector.

In order to add the termination codon and restriction enzyme NotI sitein C-terminal of the full length sequence, i.e. after Val at No. 659 inthe sequence listing, SEQ ID NO: 3 by using pBSEco-DL-2 as template, andusing the Mutagenesis kit similarly oligonucleotides having genesequences in the sequence listing, SEQ ID NO: 21 and SEQ ID NO: 18 wereset as primers, and the termination codon and NotI site were added inthe C-terminal. This vector was digested by EcoRI and NotI, and about2200 bp split gene fragment was ligated into pcDNA3, which was treatedsimilarly by restriction enzymes, to construct the expression vector.This vector was designated as pHD2F.

5) Expression Vector of FLAG Chimera Protein (HD2FLAG) of Full LengthNovel Human Delta-2

The cDNA coding chimera protein, having an amino acid sequence from No.1 to 659 in the sequence listing, SEQ ID NO: 3 and at this C-terminalthe FLAG sequence, was ligated into the expression vector pcDNA3 toprepare the expression vector.

In order to add FLAG sequence, the termination codon and restrictionenzyme NotI site in the C-terminal, by using pBSEco-DL-2 as template,oligonucleotides having gene sequences in the sequence listing, SEQ IDNO: 22 and SEQ ID NO: 18 were used as primers, and a gene coding FLAGsequence and termination codon as well as NotI site were added in theC-terminal.

This vector was digested by EcoRI and NotI, and about 2100 bp split genefragments were ligated into pcDNA3, which was treated similarly byrestriction enzyme to construct the expression vector. This vector wasdesignated as pHD2FLAG.

EXAMPLE 4

Gene Transfer of Expression Vectors Into Cells and Expression

The expression vectors prepared in Example 3 were transferred into COS-7cell (obtained from RIKEN Cell Bank, Physical and Chemical ResearchInstitute, Japan, RCB0539).

Cell culture before gene transfer was performed by culturing in D-MEM(Dulbecco modified Eagle's medium, GIBCO-BRL Inc., U.S.A.) 10% FCS. On aday before gene transfer, medium of cells was changed to set cell counts5×10⁵ cells/ml and cultured for overnight. On the day of gene transfer,cells were sedimented by centrifugation, washed twice with PBS (−) bycentrifuge and prepared to 1×10⁷ cells/ml in 1 mM MgCl₂ and PBS (−).Gene transfer was performed by electroporation using gene transferdevice Gene-pulsar (BioRad Inc., U.S.A.). The above cell suspension 500μl was collected in a cell exclusively for electroporation (0.4 cm),added expression vector 20 μg, and allowed to stand on ice for 5minutes. Thereafter, voltage was charged twice under the condition of 3μF. 450 V, and between the two charges the cell mixture was allowed tostand at room temperature for 1 minute. After 5 minutes allowed to standon ice, cells were spread in the culture medium, diameter 10 cmpreviously added 10 ml of medium, and cultured at 37° C. in 5% carbondioxide incubator.

Next day, the cultured supernatant solution was removed, washed thecells adhered to the dish twice with PBS(−) 10 ml. In cases ofexpression vectors pHD2EX, pHD2EXFLAG and pHD2EXIg, serum-free D-MEM 10ml was added and cultured for further 7 days. Cultured supernatantsolution was recovered and was replaced the buffer to PBS (−) byCentricon 30 (Amicon Inc., U.S.A.) and simultaneously the solution wasconcentrated to 10-fold to obtain cell cultured supernatant solution.

In cases of pHD2F and pHD2FLAG, medium was changed by D-MEM containing10% FCS, and cultured further 3 days to prepare cell lysate. Thus, 2×10⁶cells were suspended in the cell lysis buffer [50 mM Hepes (pH 7.5), 1%Triton X100, 10% glycerol, 4 mM EDTA, 50 μg/ml Aprotinin, 100 μMLeupeptin, 25 μM Pepstatin A and 1 mM PMSF] 200 μl, allowed to stand onice for 20 minutes and centrifuged at 14000 rpm for 20 minutes to removesupernatant solution to obtain cell lysate.

Using the thus obtained samples, expression of proteins were detected byWestern blotting.

Namely, concentrated cultured supernatants or cell lysates weresubjected to SDS-PAGE using an electrophoresis tank and polyacrylamidegel for SDS-PAGE (gradient gel 5-20%) (ACI Japan Inc., Japan) accordingto the attached instruction manual. Samples were prepared by treatmentin boiling water for 5 minutes with 2-mercaptoethanol (2-ME) forreduction, and non-reduced condition without taking the above treatment.As a markers Rainbow Marker (higher molecular weight, Amersham Inc.) wasused. Sample buffer solution and electrophoresis buffer were preparedwith reference to the attached leaflet. When the SDS-PAGE was finished,acrylamide gel was transcribed to PVDF membrane filter (BioRad Inc.,U.S.A.) using the Mini Trans Blot Cell (BioRad Inc.).

The thus prepared filter was shaken overnight at 4° C. in the Blockace(Dainippon Pharm. Co., Japan) or TBS-T [20 mM Tris, 137 mM NaCl (pH 7.6)and 0.1% Tween 20] containing 5% bovine serum albumin (Sigma Co.,U.S.A.) for blocking. Thereafter, according to the explanation of theattached leaflet of the ECL Western blotting detection system (AmershamInc., U.S.A.); anti-human Delta-2 mouse monoclonal antibody described inExample 6 or mouse monoclonal antibody Anti-FLAG M2 (Kodak Inc. U.S.A.)for FLAG chimera (HD2EXFLAG and HD2FLAG) was used as primary antibodyand peroxidase labeled anti-mouse Ig sheep antibody (Amersham Corp.,U.S.A.) was reacted as secondary antibody. In case of IgG chimera,peroxidase labeled anti-human Ig sheep antibodies (Amersham Inc.,U.S.A.) was reacted.

Reaction time for antibodies was 1 hour at room temperature, and at aninterval of each reaction, washing was performed by shaking in TBS-T atroom temperature for 10 minutes for three times. After the finalwashing, the filter was immersed in the reaction solution of ECL-Westernblotting detection system (Amersham Inc., U.S.A.) for 5 minutes, andwrapped in polyvinylidene chloride wrapping film to expose X-ray film.

As the result, in the sample with treatment of reduction, the bandsshowing a protein obtained by transfer of pHD2EX and pHD2EXFLAG weredetected a band of about 65 kD and a protein obtained by transfer ofpHD2EXIg was detected a band of about 95 kD. In the non-reduced sample,the bands showing protein obtained by transfer of pHD2EXIg were detectedslightly smeared bands at 150 kD to 200 kD, mainly about 180 kD, whichshowed about 2-fold of the reduction stage, consequently, dimer wasformed.

In these experiments, however, although cell lysate and culturedsupernatant of COS-7 cells, to which pcDNA3 vector was transferred as acontrol, were tested, no bands reacted against anti-human Delta-2 mousemonoclonal antibody, anti-FLAG antibody, and anti-human Ig antibody weredetected.

Therefore, these five expression vectors can produce the objectivepolypeptides.

EXAMPLE 5

Purification of Secretory Novel Human Delta-2 Proteins of Gene TransferCells

Cultured supernatants of COS-7 cells containing HD2EXFLAG or HD2EXIg,both of which expression was detected by the method in example 4, wereprepared in large scale, and each chimera protein was purified byaffinity column.

In case of HD2EXFLAG, 2 liter of the cultured supernatant obtained bythe method in the example 4 was passed through a column packed withAnti-FLAG M2 Affinity Gel (Eastman Kodak, U.S.A.) and the chimeraprotein was adsorbed in a column by an action of affinity between FLAGsequence of the chimera protein and Anti-FLAG antibody of the gel.Column, a disposable column (BioRad Inc., U.S.A.) with inner diameter of10 mm, was used with packing the above gel 5 ml. A circulation systemconsisting of medium bottle→column→peristaltic pump→medium bottle wasset up. The circulation was run by a flow 1 ml/min. for 72 hours.Thereafter the column was washed with PBS (−) 35 ml and eluted with 0.5M Tris-glycine (pH 3.0) 50 ml. The eluate of 25 fractions, each 2 ml,was collected into the tube, and each fraction was neutralized by 200 μlof 0.5 M Tris-HCl (pH 9.5) previously added in each tube.

The eluate fraction, each 10 μl of the secretory FLAG chimera protein,which was purified by the above method, was subjected to reductiontreatment described in Example 4. SDS-PAGE electrophoresis by 5-10%gradient polyacrylamide gel was performed. After finishing theelectrophoresis, silver staining was conducted by using Wako silverstain kit II (Wako Pure Chemicals, Japan) according to the explanationof the attached leaflet. As a result, a band of HS2EXFLAG was detectedin the eluate fractions from No. 4 to 8. Molecular weight thereof wasidentical with the result of Western blotting of anti-FLAG antibodyobtained in the example 4. Therefore, purified HD2EXFLAG was obtained.

In the IgG1Fc chimera protein, i.e. HD2EXIg, 2 liters of the culturedsupernatant was adsorbed to Protein A Sepharose column (Pharmacia Inc.,Sweden) according to the same method as of FLAG chimera protein tocollect the eluate fractions. Using a part of eluate as same as in FLAGchimera protein, a determination of the eluate fraction, identificationof the size and detection of the purity were performed by SDS-PAGEelectrophoresis and silver staining in the reduced condition. As aresult, bands were detected in the eluate fraction from No. 4 to 15. Thesize thereof is identical with the result of Western blotting usinganti-human Ig. Therefore, purified HD2EXIg was obtained.

Molecular weight of the thus purified HD2EXFLAG was further analyzed indetails by SDS-PAGE. The molecular weight was confirmed as two bands,one of which was 65.8 KD and the other was 61.7 KD. These different twobands having different molecular weights were transcribed into PVDFaccording to the method in the example 4, and ten amino acids ofN-terminal amino acid sequence were determined by amino acid sequencer(ABI Corp., U.S.A.). As a result, each amino acid sequence was identicalwith the amino acid sequence from No. 1 to 10 in the sequence listing,SEQ ID NO: 1. This result indicates that difference in molecular weightmight be due to difference in attached sugar chain. Similarly, in thepurified HD2EXIg, two bands having slightly different molecular weightwere confirmed and were thought to be due to the same reason.

EXAMPLE 6

Preparation of Antibody Recognizing Novel Human Delta-2

HD2EXFLAG, purified by the method in Example 5, was used as immunogen,and rabbits were immunized. After assaying antibody titer, whole bloodwas collected and serum was obtained. Anti-human Delta-2 rabbitpolyclonal antibody was prepared by using the Econopack serum lgGpurification kit (BioRad Inc., U.S.A.) with reference to the attachedinstruction manual.

HD2EXFLAG purified by a method described in the example 5 was used asimmunogen, and mouse monoclonal antibody was prepared according to theexplanation in the textbook. The purified HD2EXFLAG was administered inBalb/c mice (Nippon SLC CO., Japan), 10 μg/mouse, immunizedintracutaneously and subcutaneously. After second immunization,increased serum titer was confirmed by collecting bloodophthalmologically, the third immunization was performed. Subsequently,the spleens of mice were collected and fused with mouse myeloma cellsP3X63Ag8 (ATCC TIB9) using polyethylene glycol. Hybridoma was selectedby HAT medium (Immunological and Biological Research Institute, Japan),and the hybridoma strains, which produced antibody specificallyrecognizing extracellular region of novel human Delta-2 in the medium,were isolated by enzyme immunoassay. The hybridoma strains producingmouse monoclonal antibody, which specifically recognized novel humanDelta-2, were established.

The novel human anti-human Delta-2 monoclonal was purified and preparedby using Mab Trap GII (Pharmacia Inc., Sweden) according to theexplanation of the attached instruction manual from the supernatant ofthe thus established hybridoma.

Using the monoclonal antibodies, affinity column was prepared.Preparation of the affinity column was performed according to theinstruction manual attached to the CNBr activated Sephadex 4B (PharmaciaInc., Sweden). A column, 2 cm²×1 cm, containing gel 2 ml, was prepared.A concentrated solution of the supernatant of the cultured COS-7 cells,to which pHD2EX was gene transferred, was passed through the column, forwhich anti-human Delta-2 monoclonal antibody was bound, at 20 ml/hr,subsequently PBS (−) 15 ml was passed at the same flow rate and washedthe column. Finally, the product was eluted by a mixture of 0.1 M sodiumacetate and 0.5 M NaCl (pH 4.0). The eluate, each 1 ml fraction, wascollected, and was neutralized by adding 1M Tris-HCl (pH 9.1) 200 μl foreach fraction.

SDS-PAGE of each purified protein was conducted under reduced conditionaccording to the method described in the example 4, followed by silverstaining and Western blotting to estimate molecular weight. As a result,HD2EX of about 65 kD was purified from concentrated supernatant of thecultured COS-7 cells, to which pHD2EX was gene transferred.Consequently, the novel human Delta-2 protein can be purified by theaffinity column.

EXAMPLE 7

Effects of HD2EXIg to Colony Formation of Blood Undifferentiated Cells

In order to observe physiological action of HD2EXIg on bloodundifferentiated cells, CD34 positive cells were cultured in theserum-free semi solid medium in the presence of HD2EXIg and knowncytokines, and number of colony forming cells were observed.

Human umbilical cord blood or adult human normal bone marrow blood wastreated by the silica solution (Immunological and Biological ResearchInstitute, Japan) according to the attached instruction manual, then thelow density cellular fraction (<1.077 g/ml) was fractionated bydensitometric centrifugation of Ficoll pack (Pharmacia Inc., Sweden) toprepare mononuclear cells, and CD34 positive cells of human umbilicalcord blood or human normal bone marrow blood was isolated from themononuclear cells.

Separation of CD34 positive cells was performed by using Dynabeads M-450CD34 and DETACHaBEADS CD34 (Dynal Inc., Norway) according to attachedinstruction manual. After separation, the purity was measured asfollows. Cells were stained by FITC labeled CD34 antibody HPCA2(Beckton-Deckinson Inc., U.S.A.) and examined by flow-cytometer(FACSCalibur, Beckton-Deckinson, U.S.A.). Purity above 85% was confirmedfor use.

The thus isolated CD34 positive cells were suspended homogeneously toform 400 cells/ml of the medium hereinbelow, and spread in the 35 mmdish (Falcon Inc., U.S.A.), then cultured for 2 weeks in carbon dioxideincubator at 37° C. under 5% carbon dioxide, 5% oxygen, 90% nitrogen and100% humidity. The thus formed blood colonies were counted under theinvert microscope.

A medium used is α-medium (GIBCO-BRL, U.S.A.), containing 2% deionizedbovine serum albumin (BSA, Sigma, U.S.A.), 10 μg/ml human insulin(Sigma, U.S.A.), 200 μg/ml transferrin (Sigma, U.S.A.), 10⁻⁵ M2-mercaptoethanol (Nakarai Tesk Co., Japan), 160 μg/ml soybean lecithin(Sigma, U.S.A.), 96 μg/ml cholesterol (Sigma, U.S.A.) and 0.9%methylcellulose (Wako Pure Chemicals, Japan).

To the above medium, the novel human Delta-2 extracellular Ig chimeraprotein (HD2EXIg) was added to the final concentration of 1 μg/ml. Forcontrol, human IgG1 (Athens Research and Technology Inc., U.S.A.) wasadded with the same concentration in order to observe effect of IgGFcregion.

Conditions of simultaneously added cytokines were as follows: 100 ng/mlhuman SCF (Intergen Inc., U.S.A.), 10 ng/ml human IL-3 (Intergen Inc.,U.S.A.), and 100 ng/ml human IL-6 (Intergen Inc., U.S.A.).

As a result, in the control group, number of colony formation was 42±5per 400 cells, and in HD2EXIg added group, number of colony formationwas 21±3, which showed significant suppression of colony formation.Result indicates that the novel human Delta-2 of the present inventionhas an action on the blood undifferentiated cells.

EXAMPLE 8

Action of HD2EXIg on LTC-IC of Blood Undifferentiated Cells in SerumFree Liquid Culture

For confirmation of physiological action of HD2EXIg on theundifferentiated blood cells in the liquid culture, umbilical cord bloodmononuclear CD34 positive cells were cultured in serum-free medium inthe presence of HD2EXIg and known cytokines. Culture has continued for 2weeks, and at the present day on 2 weeks culture, changes of LTC-IC,which was thought to be most undifferentiated blood cells, wereconfirmed.

For comparative studies, a control experiment without adding HD2EXIg andan experiment with addition of HD1EXIg, which was IgG chimera protein(HD1EXIg) and was found to have LTC-IC activity in the similarexperiment disclosed in WO 97/19172 in the name of present inventors,were conducted. Preparation of HD1EXIg was performed according to thedescription of WO 97/19172.

Sixteen thousands and two hundreds CD34 positive cells of umbilical cordblood mononuclear cells isolated by a method described in the example 7were cultured in the following medium. Numbers of LTC-IC were counted inthe four experimental groups of the pre-culture group, HD2EXIg addedgroup, HD1EXIg added group and control group. Number of cells and numberof colony forming cells were also counted.

Culture was performed in the basal medium of α-medium, to which 2% BSA,10 μg/ml human insulin, 200 μg/ml transferrin, 40 μg/ml low densitylipoprotein, 10⁻⁵ M 2-mercaptoethanol, 100 ng/ml human SCF, 10 ng/mlhuman IL-3 and 100 ng/ml human IL-6 were added. To the HD2EXIg addedgroup, purified HD2EXIg 1 μg/ml was added; to the HD1EXIg added group,purified HD1EXIg 1 μg/ml was added; and to the control group, the abovehuman IgG1 was added. Exchange of medium was conducted twice a week withchanging half volume.

LTC-IC was measured according to a method of Sutherland et al. (Blood,74, 1563-, 1989; Proc. Natl. Acad. Sci., U.S.A., 87, 3584-, 1990). Indetails, refer to WO 97/18172 in the name of the present inventors.

Total cell counts were measured by counting numbers of living cellsmicroscopically using trypan blue (Gibco BRL, U.S.A.). Numbers of colonyforming cells were performed according to a method in the example 7using the following medium. The medium is α-medium, to which 30% fetalcalf serum (FCS, ICN Biomedical Japan, Japan), 1% BSA, 10⁻⁵ M2-mercaptroethanol, 0.9% methylcellulose (Wako Pure Chemicals, Japan),100 ng/ml human SCF, 10 ng/ml human IL-3, 100 ng/ml human IL-6, 2U/mlhuman EPO (Chugai Seiyaku Co., Japan) and 10 ng/ml human G-CSF(IntergenInc., U.S.A.).

Result is shown in Table 1.

Table 1 Effect of the human Delta-2 of the present invention on liquidculture

TABLE 1 Effect of the human Delta-2 of the present invention on liqidculture Total cell counts Colony forming cells LTC-IC Pre-culture group16200 2500 150 Post-culture group Control 445000 23000 3.3 HD1EXIg395000 16700 9.3 HD2EXIg 418000 16000 16.2

Result indicates that HD2EXIg has maintenance activity for numbers ofLTC-IC as compared with the control group. Further, the activity isstronger than HD1EXg.

EXAMPLE 9

Binding and Separation of HD2EXIg for Blood Undifferentiated Cells

Binding of purified HD2EXIg with human T cell type blood cell strainJurkat and human umbilical cord blood mononuclear CD34 positive cellswas studied. In the binding experiment, human Delta-1 chimera protein(HD1EXIg), which was found to have the similar activity by the presentinventors, was used as comparative experiment as like in the example 8.

Jurkat cells, 1×10⁶ cells, were suspended in Hank's balanced saltsolution (Gibco BRL, U.S.A.) containing 2% FCS and 10 mM Hepes. HD2EXIg,HD1EXIg or human IgG1, each 1 μg/ml, were added therein and allowed tostand at 4° C. for 3 hours. Cells were washed with the Hank's solutionby centrifugation, and PE (phycoerythrin) labeled sheep anti-human IgGmonoclonal antibody 1 μg/ml was added, then the mixture was allowed tostand in ice-cooling for 30 minutes. Thereafter, the mixture was washedtwice with the Hank's solution. Analysis was performed using the flowcytometer FACScalibur (Beckton and Dekinson, U.S.A.).

Results are shown in FIG. 2. A vertical axis indicates cell counts and ahorizontal axis indicates fluorescence intensity. The upper indicatesresult of the control HD1EXIg and the lower indicates result of HD2EXIgof the present invention. Staining with HD1EXIg or HD2EXIg is shown bysolid line and staining with human IgG1, a control group, is shown by abroken line. In all cases, binding with Jurkat cells was observed.Observing the binding activities, fluorescence intensity of HD2EXIg ofthe present invention was stronger than that of HD1EXIg of the upper.Lean fluorescence intensity of HD2EXIg was about twice-fold strongerthan that of HD1EXIg. As a result, HD2EXIg is bound with Jurkat cellsstronger than HD1EXIg.

Binding activity to the human umbilical cord blood mononuclear CD34positive cells isolated by the method described in the example 7 wasdetermined by the same staining method. In this case, staining with FITClabeled anti-human CD34 antibody HPCA-2 (Beckton and Dekinson Corp.,U.S.A.) was performed simultaneously in the second antibody labeling.Data is shown only CD34 positive, i.e. FITC positive fraction.

Result is shown in FIG. 3. Result indicates that HD2EXIg and HD1EXIgsimilarly bind with CD34 positive cells, and binding activity of HD2EXIgis two-fold stronger than that of HD1EXIg.

HD2EXIg stained cells were treated by cell sorter FACSvantage (Becktonand Dekinson Corp., U.S.A.) according to the attached instruction manualto separate cells of HD2EXIg positive fraction.

EXAMPLE 10

Effect of Cocultivation with the Novel Human Delta-2 Expression Cells onBlood Undifferentiated Cells

FLAG chimera protein expression vector pHD2FLAG of the full length novelhuman Delta-2 prepared in the example 3 was gene transferred into mousecell strain Balb3T3 (Physical and Chemical Institute, Cell DevelopmentBank RCB0005) according to a method described in the example 4 andselection was conducted by G418 (Gibco BRL Inc., U.S.A.) according tothe known method to obtain clones. The thus obtained clones wereconfirmed their expression of FLAG chimera protein of the full lengthhuman Delta-2 according to the method described in the example 4 andclones, for which expression was confirmed, were used in the followingexperiments. The clone is designated as Balb/HD2FLAG.

The umbilical cord blood mononuclear CD34 positive cells obtained by themethod in the example 7 and Balb/HD2FLAG were cocultured. Cocultivationwith Balb3T3, for which no gene transfer was made, was conducted as acontrol.

Culture conditions are:

-   -   1) Balb3T3 without gene transfer and no hematopoietic factor,    -   2) Balb/HD2FLAG and no hematopoietic factor,    -   3) Balb3T3 without gene transfer and hematopoietic factor, and    -   4) Balb/HD2FLAG and hematopoietic factor.

Medium used is α-medium added with 10% FCS and 10⁻⁵ M 2-mercaptoethanol.In the hematopoietic factor added group, 100 ng/ml human SCF, 10 ng/mlhuman IL-3 and 100 ng/ml human IL-6 were added. Cultivation is continuedfor 2 weeks and medium exchange was performed three times a week forhalf volume. Prior to cocultivation with human blood cells, previouslycultured Balb3T3 cells were irradiated with 250 KV Peak X-ray tosuppress cell growth.

Numbers of colony forming cells and LTC-IC were measured in thepre-cultivation and the experimental groups 1) to 4).

Results were as follows.

In the pre-cultivation, total cell counts were 20000 cells. Among those,colony forming cells were 3200 cells and LTC-IC was 220 cells.

In 1), colony forming cells and LTC-IC counts were very few forimpossible to measure.

In 2), numbers of colony forming cells were very few for impossible tomeasure. LTC-IC counts were 105 cells.

In 3), numbers of colony forming cells were 26500 and LTC-IC counts were90.

In 4), numbers of colony forming cells were 38000 and LTC-IC counts were120.

Analysis of breakdown of colony forming cells in 3) and 4) indicatedthat in 3), only granulocyte colony was observed, however in 4)erythroblast was also observed. Consequently, difference in number ofcolony forming cells were due to difference in number of erythroblastcolony.

As a result, Balb/HD2FLAG cell has colony forming action, especiallyerythroblast colnoy grwoth action as well as LTC-IC maintenance action.Result indicates that using the human Delta-2 expression vector of thepresent invention, cells having hematopoietic cell maintaining activitycan be produced.

EXAMPLE 11

Preparation of Material Immobilized with the Novel Human Delta-2 and ItsEffect

Sepharose gel immobilized with HD2EXIg prepared in the example 5 wasprepared. Sepharose gel used was CNBr activated Sepharose gel (PharmaciaInc., Sweden). HD2EXIg was immobilized according to the attachedinstruction manual.

The thus prepared gel and the umbilical cord blood mononuclear CD34positive cells isolated by the method in the example 7 were cultured fortwenty-four hours. Cultivation was conducted in the same medium as inthe example 10. In the control group, Sepharose gel immobilized with BSAwas prepared. After cultivation, numbers of colony forming cells weremeasured according to the method in the example 8. As a result, in thegel immobilized with HD2EXIg, numbers of colony forming cells weredecreased about 40%.

Consequently, the material, to which human Delta-2 of the presentinvention was immobilized, has an action against hematopoietic cells.

EXAMPLE 12

Effect of the Novel Human Delta-2 on Vascular Endothelial Cells

Fourth subculture of vascular endothelial cells, i.e. normal humanaortic endothelial cells and normal human lung artery endothelial cells(Kurabo Co., Japan), were used. Cells were spread 5000 cells/well in 96well-plate for cell culture (Falcon Inc., U.S.A.) at the culture ofthird subculture and were cultured in the low serum medium forendothelial cell growth (HuMedia-EG2, Kurabo Co., Japan) containinghuman recombinant EGF (Kurabo Co., Japan) 10 ng/ml and human recombinantFGF-B 5 ng/ml. At the same time, the novel human Delta-2 extracellularchimera protein (HD2EXIg) was added for final concentration of 1 μg/ml.In the comparative experiment group, same concentration of human IgG1(Athens Research and Technology Corp., U.S.A.) was added in order toobserve effect of IgGFc region. In the control group, cultivation wasconducted without adding protein except for HuMedia-EG2. Cultivation wasconducted at 37° C., under 5% carbon dioxide gas and 100% humidity for 3days, and numbers of cells were counted.

Counting for numbers of vascular endothelial cells was performed byusing NR reagent set (Kurabo Co., Japan), the principle of which wasdeveloped by Borenfreund and Purerner (Journal of Tissue Culture Methods9 (1), 7-9, 1984), i.e. neutral red method, which was applied thephenomenon that vital staining pigment neutral red(3-amino-7-dimethylamino-2-methylphenazine) can only permeate throughcell membrane of living cells to accumulate in the lysosome. Absorptionat 540 nm was measured by Immunoreader (NJ-2000, Nippon Intermed Co.,Japan). As a result, in case of aortic endothelial cells, opticaldensity (OD) in the control group was 0.18±0.02, and OD in the humanIgG1 added group was almost same grade, 0.17±0.02, however OD in theHD2EXIg added group was significantly low grade, 0.11±0.01. In case ofpulmonary artery endothelial cells, optical density (OD) in the controlgroup was 0.16±0.02, and OD in the human IgG1 added group was almostsame grade, 0.16±0.02, however OD in the HD2EXIg added group wassignificantly low grade, 0.08±0.01. These results indicate that HD2EXIgsuppresses growth of vascular endothelial cells.

EXAMPLE 13

Preparation of Drug Formulation

Each polypeptide shown in the example 5, 1 mg and human serum albumin(Midori Juji Co.) 5 mg were dissolved in distilled water 1 ml. Thesolution was aseptically passed through 0.22 μm filter forsterilization, dispensed into a vial and lyophilized to prepare drugformulation.

Effect of the Invention

The novel human Delta-2 molecule of the present invention can be usedfor effective chemicals for suppression of growth and differentiation ofundifferentiated cells such as undifferentiated blood cells, and can beused as pharmaceuticals and medical care materials.

1. An isolated DNA encoding an amino acid sequence comprising thesequence of SEQ ID NO:
 1. 2. The isolated DNA according to claim 1encoding an amino acid sequence comprising the sequence of SEQ ID NO: 2.3. The isolated DNA according to claim 2 comprising the nucleotidesequence of nucleotides 335-1854 of SEQ ID NO:
 4. 4. The isolated DNAaccording to claim 1 encoding an amino acid sequence comprising thesequence of SEQ ID NO:
 3. 5. The isolated DNA according to claim 4comprising the nucleotide sequence of nucleotides 355-2331 of SEQ ID NO:4.
 6. The isolated DNA according to claim 1 comprising the nucleotidesequence of nucleotides 355-927 of SEQ ID NO:
 4. 7. A recombinant DNAcomprising the DNA of claim 1, ligated to a vector which can express DNAin a host cell.
 8. An isolated cell transformed by the recombinant DNAof claim
 7. 9. A method comprising transforming human cells with arecombinant DNA encoding an amino acid sequence comprising the sequenceof SEQ ID NO: 1 to obtain the transformed cells of claim
 8. 10. Aprocess for the production of a polypeptide comprising an amino acidsequence of SEQ ID NO: 1, said method comprising culturing the cells ofclaim 8 under conditions in which said polypeptide is expressed, andisolating said polypeptide.